Purification and separation of sapogenins by adsorption



PURIFICATION, AND SEPARATION. OF SAPQGENINS BY nsonrrrors Monroe E.Wall, Orelan'd, and Merle: Kidder, Philadelphia, Pa., assignors tothe'United States of America as represented by the Secretary ofAgriculture No Drawing. Application July 29, 1952, Serial No. 301,614 a3 Claims, 01. zen- 2 3 9.55 (Granted under Title 35, U. s. Code 1952 t.266) A non-exclusive, irrevocable, royalty-free license in the inventionherein described, for all governmental purposes, throughout the world,with the power to grant sublicenses of the United States of America.

This invention relates to and has among its objects the purification ofcrude sapogenin mixtures and the separation of such mixtures into theirindividual components.

Sapogenins are steroidal'compounds, some of which serve as usefulprecursors for cortical and sex hormones Sapogenins are found in plantsin a combined, glycosidal form known as saponins. Upon treatment inaknown manner of crude plantextracts with strong, mineral acid,

the saponins are hydrolyzed and the sapogenins liberated. Due to theaction of the acid, many pigments and resins are formed which makeisolation, purification of the cult.

Often three or four diiferent sapogenins will ,occur in a single plantspecies. Since certain of these sapogenins are more useful than othersin hormone synthesis, it is desirable to separate them from each other.I

For the sake of convenient reference, the" more important types ofsapogenins are listedbelowin order of increasing adsorption affinity.

TABLE I Group 7.-Polyhydroxy saturated ketonicf 1V Group 8.-Polyhydroxyunsaturated ketonic f f} Adsorbents that are most useful in separatingthe above, compounds are alumina, magnesia, and silica gel; Therelatively non-polar organ sapogenin solvents that are most useful inorder of increasing polarity are hexane,

1 Z.:71 1,4os 1 Pa te nted June 21, 1955 Attirnes; derivatives of, thesapogenins may be'more easily separatedQfI'his is particularly true ofthe polyhydroxy sapogeninslisted in groups S- 8of the, table.

" Owing to thepresence of severahhydroxyl groups, these compoundsare.--;tenacious1y adsorbed. i Such, fsapog enins arejeasilyconvertedto; the corre's pondingesters; suchfas acetates, The polyhydroxyfunction is now absent: Such- 1 mixtures can beteasily; resolved and,ar'efltreatedg in the.

sampimanmf a r ps .1 4. i a,

Co poundsin'groups l'-4 o f thel tablecan-beresolved by starting 'with:benzene and gradually increasing the 'i roportionof chloroform upto 50%chloroform'in benlz ene. Compounds in 'grotips15-8 ares'eparatedbystart- T 7 ing with benzene containing'12,% alcohol and groduallyincreasing the alcoholconcentrationto by 'jvolumer Other combinations ofsolventsglisted previously mayalso T The solvent, was benzene.'The'crude' sapogenin was com I v be used to achieveth'esame etfect.-for such purposes, 1s hereby granted to theGovernment a r .Eaicample lleaves were chromatographed on 400 grams; alumina.

taminated .withj'much dark" resinous materialqQ Oh wash- 1 ing with500'ml.'benzene,'10v gramsof e'sseintially resinous materialwererecovered. .Further washing'iwithgbenzene and 1% chloroform in benzene,resulted jin reco very of, f 5 grams tigQgeninKgroupl).

eluted with l0% chloroformin benzene and 5 g'rams of of 1% chloroforminbenzene."

benzene, diethyl ether, chloroform, acetone and methyl The column isthen washed with solvent using pressure or vacuum as a means of inducingsolvent flow until the eflluent liquor has little or no solids.

more polar than the preceding one. The first solvent is slightly polar,for example, benzene with 1% chloro- Then a succession of solvents ispassed thru the column, each one :being a form. In this manner, startingwith benzene and lending with 20% alcohol in benzene, a mixture ofthevarious sapogenins found in groups1-8 of Table I' may, be sepaa rated.

sapogenins, and their separation into components difiihecogcnin (group3) .were recovered. FurtherT-washmg of the column with chloroform and 1%alcohol' 'in benzene, resulted in recovery of '7 grams resin..Onlwashingthe column with 5% 1 alcohol in benzene, 4 =gframs.rofgitogenin; (group ,containing aboutj30%*,manogenin (group 37) wererecovered. Further'f'washingl':with':10% alcohol in benzene resulted inrecovery'of 6 gramsm'anogenin containing'some .gitogenin;

A- considerable. quantity of' resin'was,irreversiblyladsorbed'onthercolumn. Hence in the example resente T some. resin is removed beforeany sapogeninsjcbmeotff. the column and-another resinous fractionremains igbe-' 7 hind after all the sapogeninshave been recovered, I

The gitogenin-manogenin' mixtures inth'efabov'e exam ple'can befurther,purified by conversionto' the acetates; followed b'yasecond passagethroughta new adsorption column.- In'this case, gitogenin acetate-iseluted, with 50% hexane inbenzene and manogenin acetateby means" \rhefollowing example showshow. two very similar jsapogenins canbeseparated; Two grams of a sapogenin mixture, dissolved. -in benz'ene,were chromatographed' on 1' SOgr'ams alumina.By-"ultra-violetadsorption analysis, the mixture was found to contain%hecogenin (group 3), and'20% 9-dehydrohecogenin (group4). 1 On elutionsuccessively'with 5, "8, 11 0, -12: and 15%, chloroform n in benzene thehecogeninfwas concentrated'in the 5 8 and 10% fraction and its 9-dehydroanalogue in the 12 and 15% fractions. v.'l-"hef l2. and l5%' fratcions'were 'rechromatographed, using [the'fsame washing solvents mentionedpreviously. Thel2 and 15% fractions con-1 tained'300 milligramsof-9-dehydrohecog'enin. The purity of the compoundwas raisedfrom 20% toWe'claini: i

l'.-A process for the purification and separation of i sapogenins ofYucca g loriosmwhichcomprises adsorbing the sapogenin mixture"onfalumina, washing the alumina with benzene and discardingtheirnpurities thus removed,

eluting tigogenin withbenzene containing about 1% ofchloroform,-eluting,further impurities with benzene con- I tainingabout'1 of alcohol, eluting gitogenin admixed with somemanog'eninbenzene containingabout' 5%.

Next, the. column was of alcohol, eluting' manogenin admixed with somegitogenin with benzene containing abouti10% alcohol, thus leaving asubstantial proportion of the original impurities adsorbed on thealumina, and recovering the sapogenins from the various eluates.

2. A process for separating a mixture of gitogenin and manogenin whichcomprises converting them to their acetates, adsorbing the acetates onalumina, eluting gitogenin acetate with benzene containing about 50% ofhexane, eluting manogenin acetate with benzene containing about 1% ofchloroform, and recovering the gitogenin and manog'enin from thesolutions of their acetates.

3. A process for separating a mixture of hecogenin and9-dehydrohecogenin which comprises dissolving the sapogenins in benzene,adsorbing them on alumina, eluting with portions of benzene containingsuccessively about 5, 8, 10, 12 and 15% of chloroform, and recoveringhecogenin from the eluates containing 5, 8 and 10% chloroform and9-dehydrohecogenin from'the eluates containing 12 and 15% of chloroform.

' References Cited in the file of this patent UNITED STATES PATENTS2,152,625 Dirscherl Apr. 4, 1939

1. A PROCESS FOR THE PURIFICATION AND SEPARATION OF SAPOGENINS OF YUCCAGLORIOSA WHICH COMPRISES ADSORBING THE SAPOGENIN MIXTURE ON ALUMINA,WASHING THE ALUMINA WITH BENZENE AND SISCARDING THE IMPURITIES THUSREMOVED, ELUTING TIGOGENIN WITH BENZENE CONTAINING AOUT 1% OFCHLOROFORM, ELUTING FURTHER IMPURITIES WITH BENZENE CONTAINING ABOUT 1%OF ALCOHOL, ELUTING GITOGENIN ADMIXED WITH SOME MANOGENIN WITH BENZENECONTAINING ABOUT 5% OF ALCOHOL, ELUTING MANOGENIN ADMIXED WITH SOMEGITOGENIN WITH BENZENE CONTAINING ABOUT 10% ALCOHOL, THUS LEAVING ASUBSTANTIAL PROPORTION OF THE ORGINAL IMPURITIES ADSORBED ON THEALUMINA, AND RECOVERING THE SAPOGENINS FROM THE VARIOUS ELUATES.